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Biosimilars: How similar is similar enough?
Peter Sebo
Biotechnologický ústav AV ČR, v.v.i.
1
What are bilogics and biosimilars ?
Biopharmaceuticals =
Protein drugs produced using live organisms
Biodrugs
Biologicals, Biologics
Biosimilars (EMEA)
Follow – on biologics – FOB (FDA)
2Poděkování: doc. M. Fusek
3
Size matters
Erytropoetin
Atorvastatin
courtessy: doc. M. Fusek
Biologics versus conventional drugs
- production systems – Live organisms used
- molecular wight of the active comnpound
- different analytics used
- different formulation
- potential immunogenicity risk
- Local versus systemic action – selectiveness?
- Legislative obstacles/biosimilars – EMEA guidelines
4
5
Total sales and sales growth trend in the US biotech market for biologic drugs (2003–2008)
6
What's fueling the biotech engine—2008
Saurabh Aggarwal
Nature Biotechnology 27, 987 - 993 (2009)
Top companies that comprise the majority of sales of biologic drugs in 2008.
7
8
Very attractive business:
1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002
in 2006 biodrugs represented 33 % of newly approved drugs
Biologicsbiosimilars
follow-on biologics
The issues…
Presentation title Date
11
Main risk =Imune response
• EPREX – Pure Red Cell Aplasia (PRCA) = anemia
• 1 per 5000 patints developed antibodies against EPO
• due to extractables form vial lids in the presence of polysorbate -adjuvans.
• Omnitrope® (somatropin) - Genotropin (Pfizer)57% of patients developed antibodies due to prsence of residual host protein contamination serving as adjuvant
• Purification redeveloped
• Phase III trial repeated
• Saspproved by EMEA, FDA, Australia
Approved Biosimilars - FOB
Name INN original approval
Omnitrope (San) Somatotropin Genotropin (Pf) 12 Duben 2006
Valtropin (Biopartners) Somatotropin Humatrope (EL) 24 Duben 2006
Binocrit (San) Epoetin alfa Eprex (Jan-Cil) 28 Srpen 2007
Epoetin Alfa Hexal (Hex) Epoetin alfa Eprex (Jan-Cil) 28 Srpen 2007
Apseamed (Medice) Epoetin alfa Eprex (Jan-Cil) 28 Srpen 2007
Retacrit (Hospira) Epoetin zeta Eprex (Jan-Cil) 18 Prosinec 2007
Silapo (Stada) Epoetin zeta Eprex (Jan-Cil) 18 Prosinec 2007
Ratigrastim (RatioPharm) Filigrastim Neupogen (Amg) 21 Únor 2008
Biograstim (CT Arzenm) Filigrastim Neupogen (Amg) 21 Únor 2008
Tevagrastim (Teva) Filigrastim Neupogen (Amg) 21 Únor 2008
Filgrastim Ratiopharm Filigrastim Neupogen (Amg) 21 Únor 2008
Future Global player – India• No. 2 producer of pediatric vaccines, No. 4 in drug manufacturing…
• big pharma uses it for outsourcing of R&D and production
• Úpatents prior to 1995 are not respected -1995 patenty =
• 48 biosimilars produced in India in 2008…
• Biosimilars 30-40% cheaper – sales grow
• Biosimilars – 35% - 65% of Indian markert of biologicals
• Qualified workforce - R&D costs 60% cheaper and 35% chepaer than in USA
• Contract research and manufacturing services – 1 mld. USD in 2010
• Indian companies buy EU biopharma firms to penetrate EU markets
• 37% of filed Drug master files in 2007 was from India …
• 130 indian biopharma companies…
• in 2006 sales of $1,5 bil. USD – CAGR 27%...
• 300 milions of Indian can afford expensive treatments - 11th marklet size
• EMEA-certifiied manufacturing of Inmtas Biopharmaceuticals Ltd…13
Will biosimilars really make it?
• The segment of the bipological market grows > 200 products on the market, >300 trials ongoing…
• 50% of pharma market by 2010 are biologicals?• Biologicals sales grew 6-fold in a decade …• The older population needs more expensive drugs …• The fastest growinhg market are antibodies (>$75 B)• >25% of drugs in the pipeline are biologicals• Unsaturated market / the producing capacity is not big
enoughnasycen – nedostatečné výrobní kapacity…• 7 patents on 8 key markets expire worth 12 bil. USD• 30% proice reduction and 30-40% market penetration expected• EU could save 20% of costs using biosimilars = 1,6 mld €/year• Much less competition then on generics – complicated
production…14
However, one should not get armed for the past war….Nowadays only a new improved generation of
biosimlars makes sense…
Big pharma biosimilar divisions ready to enter market…
Differ by application route, formulation, glycoprofile, efficay, stability etc.…..
15
Why biosimilars cannot be that much cheaper than original drugs
16
R&D costs and clinical trials plus marketing and market substitution costs high
Problems of biosimilars
• Much higher R&D costs than conventional generics
• Immunofgenicity risk
• Proving identity to original drugs difficult
• Higher production costs + clinical trials needed
• Biocomparability, PK, PD
• High marketing aand market substitution costs
• Much smaller margin in % of „biogenerics“
– Possible „discount“ 10 – 40 % - But how will originator react?
• High societal need – 30 % cheaper mAb is a lot of money
17
Presentation title Slide no 18 Date
The Basics of Biotech Manufacturing Processes and the Starting Point for Biosimilars
DNA Vector Cloning into
DNA Vector
Large-Scale Fermentation
Downstreaming
Formulation
Transfer into
Host Cell,
Expression
e.g., bacterial or mammalian cell
Maybe the same
genetic sequence
(Probably)
a different
DNA vector
Different recombinant cell
system
A different
fermentation
process
A different
purification
protocol
Different
in-process
controls
Maybe a
different
formulation
Presentation title Slide no 19 Date
An Entirely New Manufacturing Process – Innovator or Follow-on Lead to Potential Quality, Efficacy and Safety Issues
DNA Vector Cloning into
DNA Vector
Large-Scale Fermentation
Downstreaming
Formulation
Transfer into
Host Cell,
Expression
e.g., bacterial or mammalian cell
Maybe the same
genetic sequence
(Probably)
a different
DNA vector
Different recombinant cell
system
A different
fermentation
process
A different
purification
protocol
Different
in-process
controls
Maybe a
different
formulation
Clinical Studies are always required for significant risk process changes to demonstrate absence of clinically meaningful differences
Change
filter
supplier
Move
equipment
different part
of facility
Move
manufacturing
to new facility
New cell line
New Process
New Formulation
Scale-up
Manufacturing
Manufacturing Change
• Low risk• Frequent
• Supported by:
Analytical and
Process Data
• Highest risk• Rare
• Extensive Data:
Analytical, Process
and Human data
Follow-on Biologics
New:
•DNA?
•Cell line
•Process
Technology?
•Fermentation
process
•Purification process
•Analytics
•Facilities
•Formulation?
•And - no history
2nd generation innovator processes – and for FOB’sAppropriate human study to demonstrate the equivalence of the efficacy and
safety (including immunogenicity) of the product
Character of Change
Presentation title Slide no 21 Date
Omnitrope® (somatropin)Reference product: Genotropin (Pfizer)
• Early version of the product: 57% of patients developed antibodies against Omnitrope
– Problem was residual host-cell protein
• Re-developed purification process
• Conducted a second phase 3 study
• Antibody levels reduced
• Approved by EMEA, FDA and Australia
Clinical studies evaluating efficacy and safety required to identify and
exclude clinically meaningful difference
Presentation title Slide no 22 Date
Alpheon (alpha interpheron)Reference product: Roferon (Roche)
• Differences in impurity profile observed• Key Clinical Data
– PK (3 studies): supra-bioavilability/comparable/inconclusive
– PD (2 studies): PD equivalence/no PD equivalence
– Safety&Efficacy:• Clinically and statistically significant difference in virological
relapse rate found: more patients on Alpheon had relapse
• Different rate of adverse events and laboratory-related events judged as clinically relevant
Clinical studies evaluating efficacy and safety demonstrated clinically meaningful
differences leading to Rejection by EMEA
Presentation title Slide no 23 Date
Conclusion
• Biosimilars will be similar - not the same as innovator
• Characterization doesn’t tell the whole story – and chemical differences matter!
• Only way to exclude clinically meaningful differences in efficacy, safety and immunogenic potential – is from clinical data
• Product class specific guidance will ensure consistency
Presentation title Slide no 24 Date
There are no definitive technologies currently available to predict how the human immune system will respond to a product
• Immune responses to protein products can be extremely serious or life-threatening
• Immunogenicity is a significant characteristic of any protein:
– Must be included in overall comparability exercise
– Immunogenicity can only be determined in human studies…
On the other hand…
• Process can be reverse-engineered to someextent
• Several biosimilars already approved
• EMEA Guidelines available
• Some savings in clinical trials possible… refering to originator product
25
Proving identity: Are cutting edge technologies
getting us there?
dogma or propaganda?
• Comparison is possible only to marketed products…
• No in-process details or batches available from innovators…
• process = product - a dogma used as argument…
• Do laws of chemistry and physcis apply to biosimilars, too?
• Proteins are certainly not rigid and show structural dynamics and modification heterogeneity, but …
• Proteins too are chemical compounds, albeit complex…
27
Process equals product?Case study: Hen egg white lysozyme
28
Overal view Some flexibility/differences
in loops observed
Different crystal forms from different processes• 193L P 43 21 1 1.3Å• 2VB1 P 1 0.65Å• 1AKI P 21 21 21 1.5 Å• 1PS5 C 2 2.0 Å• 1VDP P 21 1.7 Å• Different chicken on differing diet in different countries…
29
Larger protein case:Coagulation factor VIII
• 2R7E P 41 21 2 3.7 Å
• 3CDZ P 41 21 2 3.98 Å
A very large protein crystallized in the same space group by different teams resolution not terrific but no great structure differences seen either. Loops may be poorly defined
The anti-dogma
• X-ray crystallography not the simplest and fastest …
• Cutting-edge Fourier transform mass spectrometry analytical methods and NMR fingerprinting are a breakthrough…
• Exact protein sequence confirmation not an issue
• Assessment of sample modifications not an issue
• Sample similarity (microheterogeineity pattern) assessment possible
• Can we start asking ‘how similar is similar’?
• Proving ‘chemical identity’ or protein and contaminant content (di-)similarity starts being really possible
• Very realistic in 5 years from now
30
The true question?
• The cutting-edge FT MS analytics will allow to optimize the process for biosimilars to yield an ‘identical’ protein product in terms of chemistry – how similar is similar enough?– Combination of ESI, ECD, MALDI approaches allows determining
chemical structure of posttranslational modifications and assessing of sample composition in terms of microheterogeneity
– Structure fingerprinting possible
• Biological activity, pharmacodynamics and equivalence testable
• Massive R&D investment for biosimilars required anyway…
• Is it faster and more cost-effective to make the biosimilarprocess yielding an ‘identical’ protein product or is it faster to go through the full clinical testing and registration procedure?
31
NMR fingerprinting of 3-D structure of unlabeled proteins as QC method…
Anal Chem. 2008 Apr 1;80(7):2623-7
Assessment of the three-dimensional structure of recombinant protein therapeutics by NMR fingerprinting: demonstration on recombinant human granulocyte macrophage-colony stimulation factor.
• Aubin Y, Gingras G, Sauvé S.
• We describe a simple, powerful, and robust NMR-based method that has the potential to greatly impact the characterization of recombinant protein therapeutics. The method
ascertains the bioactive conformational identity of recombinant human granulocyte macrophage-colony stimulation factor (rhGM-CSF) produced in Streptomyces lividans versus Escherichia coli by overlaying their 2D 1H,15N HSQC correlation spectra. An identical match of all resonances implies that
rhGM-CSF from both processes share indistinguishable conformations that correlate with in vitro activity. The result of this method is unique among existing
methods. It can detect and quantify the active ingredient. Moreover it provides a complete assessment of the conformation with high sensitivity to minor structural changes
32
No effect of excipients observed…
Used 700 MHz instrument –more performing instruments are in the pipeline….
Solve structure of reference product – compare fingerprints in process…
Comparison of active substancesanalytical scheme for active protein substance – start with basic properties
Sample
Charge
IEX, IEFMW
SEC, SDS-PAGE
(non- and reducing)
Hydrophobicity
RP, HIC Accurate MW
(LC-)MS:
ESI-FTMS
ESI-qToF2D PAGE / HPLC
Poor MW accuracy but provides
information about HMW forms
Highly accurate (sub- / ppm)
directly point on modifications and
isoforms. Still not routine for aggregates or
non-covalent oligomers
Information about number
of isoforms and/or approximate
MW
NMR
fingerprinting
Shape, structuration, oligomerization
centrifugation
gross shape,
conformational
changes
Dynamic light
scattering –
presence of
oligomers/aggrega
tion
Circular dichroism
far UV (190-250nm) –
secondary structure elements,
near UV (250-350nm) – some
aspects of tertiary structure
(follow aromatic amino acids
and SS bonds)
Fluorescence (Trp)
X-ray
full structure
Primary sequence / modifications
Unmodified protein – sequence by accurate mass not an issue anymore
Modified proteins – accurate mass point on the extent of modification
MS/MS on the entire intact protein (top-down)
+ link between selected isoform and its modifications preserved
- limitations – MW, precursor isolation
MS/MS of peptides from enzymatic digestion (bottom-up)
+ MW of the protein no longer limiting
- more difficult to link modification to parent molecule – specific cases where differentially modified isoforms exist
Glycation – Brady LJ – AnalChem 2007
Gadgil HS – AnalBiochem 2006
cutting-edge FT MS
Current limits - to be significantly improved in 3-5 years:
Dynamic range - 2-4 orders of magnitude
Sensitivity – sub-fmol
Resolving power – routinely 200,000 but much higher possible
Accuracy – sub-ppm / tens of ppb
instrument cost 2-3 M Euro
Required great guys…
SoloukiT AnalChem 1997
Selected modifications found on protein pharmaceuticals
• N-/O-glycosylation
• Pyro-Glu, Asn/Gln deamidation, isoAspartate
• Disulfide bridges
• 4-carboxyglutamate
• Sulfation
• Phosphorylation
• Glycation
• Oxidation
• PEGylation
Sample
Example work-flow for IgG characterization
digestion
Reduction/(alkylation)
Deglycosylation
(EndoH, PNGaseF)
LC-MS(/MS)-sequence coverage
-glycopeptides (type
and site of attachment)
LC-MALDI
iontrap (CID, ETD)
FTMS (CID, ECD)
Deglycosylation
(EndoH, PNGaseF)
LC-MS(/MS)
-MW after degly
to asses on
other modifications
ESI-qToF
ESI-FTMS
LC-MS(/MS)-sequence coverage
-confirmation of
glycosylation and
other PTMs
LC-MALDI
iontrap (CID, ETD)
FTMS (CID, ECD)
Sequencing of released glycans
MS(/MS)
HPAEC-PAD + MS
NMR
Glycosidases+MS
LC-MS(/MS)
-disulfide bonding
FTMS
digestion
TopDown
MS, MS/MSFT-MS (CID, EXC,IRMPD,…)
Process optimization case study
tuning the fatty-acylation…
5 15 25 35 Time [min]
Extremely large potential biopharmaceutical
(1706 aa residues)
PTMs consisting of palmitoylation
HPLC of trypsin digest
Process optimization case study
tuning the fatty-acylation…
Sequence coverage from a single LC/FT MS analytical run 94%
A mutation detected V-->L
Disulphides unambigously assigned
Acylation determined modified lysine 1 = 58% palmitoleate, 42% palmitate
modified lysine 2 = 37% palmitoleate, 43% palmitate
Tuning protein acylation in process:Extracted ion chromatograms from LC/FT MS
K1069
K946
unmodified C16:1
C16:1
C16:0
C16:0
unmodified
C16:1
C16:1
C16:0
C16:0
K1069
K946
unmodified
unmodified
Inappropriate process
1ASNSLQYVNVQVKDIEADLQHGVDESYTLDVEEDSDTITINAETVWGALHAFTTLQQLV
60ISDGHGGLIIEEPVNIKDSPLYPYRGIMLDTGRNFVSLPKIFEQLEGMSLSKLNVLHWH
119IDDAQSWPIWVDVYPEMVKDAYSPHEIYSRNDVRNIVNYARARGIRVIPEIDMPSHS
176SSGWKQVDPEMVTCTDSWWSNDDWPLHTAVEPNPGQLDIIYNKTYEVVGNVYKEL
231SDIFPDHWFHVGGDEIQPNCFNFSTHVTKWFAEDPSRTYHDLAQYWVDHAVPIFQ
286NYSQERRLVMWEDIALSADNAHDVPKNIVMQSWNNGLEYISNLTARGYDVIVSSSD
342FLYLDCGHGGFVTNDPRYNVMANPDANTPNFNYGGNGGSWCAPYKTWQRIYDYD
396FTLNLTETQAKHIIGATAPLWGEQVDDINVSSMFWPRAAALAELVWSGNRDANGNK
452RTTEMTQRILNFREYLVANGVQAQALVPKYCLQHPHACDLYRNQAAIQ
Disulfide Bridges:b-N-Acetylhexosaminidase
Six cysteine residues at positions: 189, 250, 347, 382, 482 and 489.
Used protease digestion sites underlined and in colour
Strategy
• Non-reducing SDS-PAGE
• In-gel deglycosylation (PNGaseF) and digestion(Trypsin and Asp-N).
• Capillary LC-FTMS (C18, 0.1x150 mm, 3mm, 200A,APEX-Q 9.4T).
• Data reduction (Averagine, THRASH and Z-score algorithms).
• Data interpretation (Links).
Deconvoluted spectra of cystine peptides from β-N-Acetylhexosaminidase (A. oryzae). (a) Asp-N cystine peptide Cys189/Cys250
at m/z 1857.7348. (b) Tryptic cystine peptide Cys347/Cys382 at m/z 5952.5998 and its oxidized form. (c) intrachain tryptic
cystine peptide Cys482/Cys489 at m/z 1616.7095. All peptides were measured with the error below 1.0 ppm. (d) The contribution
of 34S visible to the third isotope – I.) experimental data - cystine peptide Cys482/Cys489 at m/z 540.2401 (z = 3); II.) theoretical
spectrum of the same peptide; III.) theoretical spectrum of a peptide (SSDFLYLDCGHGGFV) which fits into the window (3ppm) of
accurate mass search, but has only one sulfur.
All 3 disulphides readily identified
The accuracy of FT MS is the key…
S-S Peptide(s) Exp. Theor. Error
1-2 183-190,244-251 1857.7348 1857.7344 0.22 ppm
183-190,244-251 ox. M 1873.7300 1873.7292 0.44 ppm
170-190,244-251 3284.3680 3284.3645 1.07 ppm
170-190,244-251,ox. M 3300.3573 3300.3594 0.67 ppm
3-4 332-358,359-386 5952.6206 5952.6069 2.30 ppm
332-358,359-386 ox. M 5968.6111 5968.6016 1.59 ppm
5-6 481-493+1S_S 1616.7095 1616.7102 0.45 ppm
S-S Linkages
1ASNSLQYVNVQVKDIEADLQHGVDESYTLDVEEDSDTITINAETVWGALHAFTTLQQLV
60ISDGHGGLIIEEPVNIKDSPLYPYRGIMLDTGRNFVSLPKIFEQLEGMSLSKLNVLHWH
119IDDAQSWPIWVDVYPEMVKDAYSPHEIYSRNDVRNIVNYARARGIRVIPEIDMPSHS
176SSGWKQVDPEMVTCTDSWWSNDDWPLHTAVEPNPGQLDIIYNKTYEVVGNVYKEL
231SDIFPDHWFHVGGDEIQPNCFNFSTHVTKWFAEDPSRTYHDLAQYWVDHAVPIFQ
286NYSQERRLVMWEDIALSADNAHDVPKNIVMQSWNNGLEYISNLTARGYDVIVSSSD
342FLYLDCGHGGFVTNDPRYNVMANPDANTPNFNYGGNGGSWCAPYKTWQRIYDYD
396FTLNLTETQAKHIIGATAPLWGEQVDDINVSSMFWPRAAALAELVWSGNRDANGNK
452RTTEMTQRILNFREYLVANGVQAQALVPKYCLQHPHACDLYRNQAAIQ
AAKAPFKIDFEVRRGESKDDLSPEDDSN
PRFVKRDGSLDMTLTNKQTFYMATLKIG
SNEDENRVLVDTGSSDLWVMSHDLKCV
SAPNSKRNERSFGHGTGVKLNERELMQ
KRKNLYQPSRTIETDEEKEASEKIHNKL
FGFGSIYSTVYITEGPGAYSTFSPFVGTE
GGSGGSGGSNTCTSYGSFNTENSDTFK
KNNTNDFEIQYADDTSAIGIWGYDDVTIS
NVTVKDLSFAIANETSSDVGVLGIGLPGL
EVTTQYGYTYQNLPLKLKADGIIAKSLYS
LYLNTADAKAGSILFGAIDHAKYQGDLV
TVKMMRTYSQISYPVRIQVPVSKIDVES
SSGSTTNILSSTTGVVLDTGSTLSYVFSD
TLQSLGKALNGQYSNSVGAYVVNCNLA
DSSRTVDIEFGGNKTIKVPISDLVLQASK
STCILGVMQQSSSSSYMLFGDNILRSAYI
VYDLDDYEVSLAQVSYTNKESIEVIGA
IKLDFNKVSTPSKYTKRDALPMPLIND
KILYTTELEIGSNKDKVSVSIDTGSYDL
WVMSNDAVCYKVSEFQTEGAPQLPDI
FNDIDQDYSCTFNGTYNSKSSKTFKN
TSEDFSIGYVDGSAAQGVWGYDSVQF
GQYGVTGLKIGIANRSSVSDGILGIGIA
NGYDNFPVLLQKQGLINKIAYSVYLNS
SNSTTGTILFGAIDHAKYKGALSTVPV
DSKSQLSVNVTNLKTKNGNVASGGHS
ILLDTGSTFSIFPDEWIDALGHSLDATY
DEDESVYEIECDGYDEHFFGFSIGDSD
FSVPIQDLKTEKDGQCYLAIMSNSVIG
GGGILFGDDILRQIYLVYDLQDMTISVA
PVVYTEDEDIEEIL
SAP 9 SAP 10
Rapid characterization of protein glycosylation at multiple sites
Strategy
• Protein reduction (TCEP) and alkylation (IAA)
• Enzymatic proteolysis (Trypsin and Asp-N)
• Capillary LC-FTMS (C18, 0.1x150 mm, 3mm, 200A, APEX-Q 9.4T)
• Data reduction (Averagine, THRASH and Z-score algorithms) and interpretation (GlycoS)
LC-FTMS 2D Plot of Sap10 Trypsin Digest and Spectra of Selected Glycopeptides.
GlycoS
SAP10 glycosylation – LC-MS/MS
Sample
microheterogeneity
Assessment
How similar
is similar enough…
Single hexose peak m/z spacing
SAP10 glycosylation – LC-MS/MS
Single hexose peak m/z spacing
Results
Generic therapeutic antibodies
Targeted biological - antibody drugs against
cancer, pain, arthritis, osteoporosis
50% of current biologicals market?
Robust
nowadays rather easy to produce
minimal postrranslational modification (PTM)
Straightforward analytics of PTM
probably the easiest to copy…
Can be produced in glycooptimized cells
established structure characterization for Fab
and Fc fragments
Take home message:
• Tremendous improvement and steady advance of analytical instrumentation makes in-process comparisons of chemical identity (similarity) of biosimilars and original products realistic
• The nebula of isoforms of original and similar molecules compared…
• Matter of time and cost-effcetiveness
• Biosimilars require substantial R&D effort anyway…
• Full clinical testing of biosimilars, can be lengthy (not only costly) due to limited numbers of patients
• Advanced analytics require costly equipments and great guys to operate them… - partner with academia!
54
Petr Man Petr Novákhttp://ms.biomed.cas.cz/equipment.php
Institute of Microbiology AS CR, v.v.i.
Prague, CR
Acknowledgements: